Method for measuring amount of residual benzyl alcohol, and use thereof

Abstract

Provided is a method for measuring the amount of residual benzyl alcohol in a sample, in particular a method for measuring the amount of residual benzyl alcohol in a process sample or a biological product stock solution of a biological product, comprising extracting benzyl alcohol in a sample by using a polar organic solvent, and measuring the amount of residual benzyl alcohol in an extraction solution by means of chromatography. Also provided is a use of the method in measurement of the amount of residual benzyl alcohol in a process sample or a biological product stock solution of a biological product.

Description

Method for determining benzyl alcohol residual amount and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, in particular to the establishment of a method for determining benzyl alcohol residual amount and the application thereof. BACKGROUND

[0002] Benzyl alcohol, also known as benzyl alcohol, is an aromatic alcohol commonly used as a preservative and fragrance in food and cosmetics, and also used as a medical preservative. Benzyl alcohol has anesthetic effect and stimulates the eyes, upper respiratory tract and skin, causes headache, nausea, vomiting, gastrointestinal irritation, convulsions and coma after ingestion, and poses certain hazards to human health. Benzyl alcohol is often used to preserve purification columns in the purification process of biological products (such as proteins), and if not thoroughly cleaned, it may result in a certain amount of benzyl alcohol remaining in the final product, thereby affecting the safety of biological product medication. Therefore, benzyl alcohol residual amount is an important indicator for evaluating drug safety.

[0003] However, the current literature reports on benzyl alcohol detection methods mainly involve samples such as cosmetics, tobacco products, traditional Chinese medicinal materials, food, toys, etc., and the sample pretreatment methods mainly include solvent dissolution / dilution, static headspace (SHS), headspace solid phase microextraction (HS-SPME), liquid-liquid extraction (LLE), solid phase extraction (SPE), etc. There are few reports on the detection method of benzyl alcohol content in biological products in the prior art, and due to the differences in sample matrix, the detection method of benzyl alcohol in other samples is not suitable for the determination of benzyl alcohol residual amount in biological products. In order to ensure the safety of biological product medication, it is urgent to develop a method for determining benzyl alcohol residual amount suitable for biological products. SUMMARY

[0004] The present application provides a method for determining benzyl alcohol residual amount, which uses chromatographic technology, and develops a simple and accurate method for determining benzyl alcohol residual amount in biological product samples based on the physicochemical properties of benzyl alcohol and the matrix characteristics of the samples.

[0005] In some embodiments, the present application provides a method for determining benzyl alcohol residual amount in a sample, which comprises: extracting benzyl alcohol in the sample with a polar organic solvent, and determining the residual amount of benzyl alcohol in the extraction solution by chromatography, wherein the sample is a biological product sample. In some embodiments, the sample is a process sample of a biological product. In some embodiments, the sample is a bulk biological product.

[0006] In some embodiments, the process is a purification process. In some embodiments, the process is selected from one or more of affinity chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, complex mode chromatography, and hydroxyapatite chromatography. In some embodiments, the process is selected from one or more of primary purification, intermediate purification, fine purification, and ultrafiltration.

[0007] In some embodiments, the biological product is a protein-containing biological product. In some embodiments, the biological product is selected from one or more of a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a recombinant protein, a tagged fusion protein, an antibody drug conjugate product. In some embodiments, the biological product is an antibody-based biological product. In some embodiments, the biological product is a therapeutic antibody-based biological product. In some embodiments, the biological product is a monoclonal antibody, a multispecific antibody, or an antibody drug conjugate product. In some embodiments, the biological product is selected from one or more of a chimeric antibody, a murine antibody, a primatized antibody, a humanized antibody, or a fully human antibody. In some embodiments, the biological product is selected from one or more of ustekinumab, golimumab, tocilizumab, bevacizumab, dupilumab, or BAT4306F.

[0008] In some embodiments, the sample is a protein-containing biological product purification process sample. In some embodiments, the sample is a protein-containing biological product bulk. In some embodiments, the sample is an antibody-based biological product purification process sample. In some embodiments, the sample is an antibody-based biological product bulk. In some embodiments, the sample is a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a recombinant protein, a tagged fusion protein, an antibody drug conjugate product purification process sample. In some embodiments, the sample is a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a recombinant protein, a tagged fusion protein, a chimeric antibody, an antibody drug conjugate product antibody bulk. In some embodiments, the sample is a chimeric antibody, a murine antibody, a primatized antibody, a humanized antibody, or a fully human antibody product purification process sample. In some embodiments, the sample is a chimeric antibody, a murine antibody, a primatized antibody, a humanized antibody, or a fully human antibody product antibody bulk. In some embodiments, the sample is a ustekinumab, golimumab, tocilizumab, bevacizumab, dupilumab, or BAT4306F antibody product purification process sample. In some embodiments, the sample is a ustekinumab, golimumab, tocilizumab, bevacizumab, dupilumab, or BAT4306F antibody product antibody bulk.

[0009] In some embodiments, the process samples or bulk biologic of the biologic is stored in a buffer, and the buffer formulation is selected from the group consisting of histidine, histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, citric acid, sodium citrate, methionine, and acetic acid. In some embodiments, the buffer formulation comprises histidine and histidine hydrochloride. In some embodiments, the buffer formulation comprises disodium hydrogen phosphate and sodium dihydrogen phosphate. In some embodiments, the buffer formulation comprises citric acid, sodium citrate, and methionine. In some embodiments, the buffer formulation comprises histidine and acetic acid.

[0010] In some embodiments, the polar organic solvent is dichloromethane, ethyl acetate, or acetonitrile. In some embodiments, the polar organic solvent is dichloromethane.

[0011] In some embodiments, the chromatography is gas chromatography-mass spectrometry.

[0012] In some embodiments, the present application provides a method for determining the residual amount of benzyl alcohol in a sample, comprising: extracting benzyl alcohol in the sample with dichloromethane, and determining the residual amount of benzyl alcohol in the extract solution by gas chromatography-mass spectrometry.

[0013] The present method can be used to measure a wide range of protein content of biologic samples. In some embodiments, the protein concentration in the biologic sample is between 1 mg / mL and 300 mg / mL. In some embodiments, the protein concentration in the biologic sample is between 1 mg / mL and 200 mg / mL. In some embodiments, the protein concentration in the biologic sample is between 1 mg / mL and 150 mg / mL. In some embodiments, the protein concentration in the biologic sample is no more than 60 mg / mL. In some embodiments, the protein concentration in the biologic sample is no more than 40 mg / mL. In some embodiments, the protein concentration in the biologic sample is between 1 mg / mL and 60 mg / mL. In some embodiments, the protein concentration in the biologic sample is between 1 mg / mL and 40 mg / mL. In some embodiments, if the protein concentration in the biologic sample is higher than 60 mg / mL, the sample is diluted to a protein concentration no more than 60 mg / mL with ultrapure water before the benzyl alcohol in the sample is extracted with the polar organic solvent. In some embodiments, if the protein concentration in the biologic sample is higher than 40 mg / mL, the sample is diluted to a protein concentration no more than 40 mg / mL with ultrapure water before the benzyl alcohol in the sample is extracted with the polar organic solvent. In some embodiments, the protein concentration after dilution is no less than 1 mg / mL. In some embodiments, the protein concentration after dilution is no less than 5 mg / mL. In some embodiments, the protein concentration after dilution is no less than 10 mg / mL.

[0014] The concentration of benzyl alcohol in the extraction solution can be determined by conventional methods, and the residual amount of benzyl alcohol in the sample can be calculated by conventional methods based on the determined concentration of benzyl alcohol in the extraction solution. In some embodiments, the residual amount of benzyl alcohol is determined by calculating the concentration of benzyl alcohol in the extraction solution by external standard method, and further calculating the residual amount of benzyl alcohol in the sample. In some embodiments, the residual amount of benzyl alcohol is determined by calculating the concentration of benzyl alcohol in the extraction solution by external standard method, and further calculating the residual amount of benzyl alcohol based on the ratio of the concentration of benzyl alcohol to the concentration of protein in the sample.

[0015] In some embodiments, the concentration of benzyl alcohol in the extraction solution is calculated by external standard method, and the residual amount of benzyl alcohol in the sample is further calculated.

[0016] In some embodiments, the residual amount of benzyl alcohol in the sample is calculated according to the following formula:

[0017] wherein,

[0018] C SA : the measured value of the concentration of benzyl alcohol in the extraction solution (ng / mL);

[0019] C mAb : the concentration of protein in the sample (mg / mL);

[0020] DF: the dilution factor of the extraction solution, which is determined by the actual preparation process of the solution.

[0021] In some embodiments, the measured value of the concentration of benzyl alcohol in the extraction solution is calculated by standard curve method. In some embodiments, calculating the measured value of the concentration of benzyl alcohol in the extraction solution comprises preparing a standard curve with the concentration of benzyl alcohol of benzyl alcohol standard solution at different concentration gradients as the abscissa (X) and the corresponding peak area as the ordinate (Y), obtaining a linear regression equation; and substituting the peak area of benzyl alcohol of the extraction solution into the linear regression equation to calculate the measured value of the concentration of benzyl alcohol in the extraction solution (ng / mL).

[0022] In some embodiments, the present application provides a method for determining the residual amount of benzyl alcohol in a sample, comprising the following steps:

[0023] Step 1, if the concentration of protein in the biological product sample is higher than 40 mg / mL, dilute the sample with ultrapure water to a protein concentration of not higher than 40 mg / mL;

[0024] Step 2, take the sample of step 1, add a polar organic solvent, invert several times, centrifuge, take out the organic phase, and optionally repeat the above operation, combine the organic phases, mix well to obtain an extraction solution for determination;

[0025] Step 3, the extraction solution is detected by chromatography, and a chromatogram is recorded;

[0026] Step 4, the concentration of benzyl alcohol in the extraction solution is calculated by external standard method, and the residual amount of benzyl alcohol in the sample is further calculated, and the specific calculation formula is as follows:

[0027] wherein,

[0028] C SA : the measured value of the concentration of benzyl alcohol in the extraction solution (ng / mL);

[0029] C mAb : the concentration of protein in the sample (mg / mL);

[0030] DF: the dilution multiple of the extraction solution, which is determined by the actual preparation process of the solution.

[0031] In some embodiments, the measured value of the concentration of benzyl alcohol in the extraction solution is calculated by standard curve method. In some embodiments, calculating the measured value of the concentration of benzyl alcohol in the extraction solution comprises drawing a standard curve with the benzyl alcohol concentration of benzyl alcohol standard solution with different concentration gradients as the abscissa (X) and the corresponding peak area as the ordinate (Y) to obtain a linear regression equation; and substituting the benzyl alcohol peak area of the extraction solution into the linear regression equation to calculate the measured value of the concentration of benzyl alcohol in the extraction solution (ng / mL).

[0032] In some embodiments, the polar organic solvent is difficult to dissolve in water. In some embodiments, the polar organic solvent is not miscible with water. In some embodiments, the polar organic solvent is dichloromethane, ethyl acetate or acetonitrile. In some embodiments, the polar organic solvent is dichloromethane.

[0033] In some embodiments, the chromatography column used in the chromatography is a non-polar or weakly polar chromatography column. In some embodiments, the chromatography column used in the chromatography is a non-polar or weakly polar polysiloxane capillary chromatography column. In some embodiments, the chromatography column used in the chromatography is a (5%-phenyl)-methyl polysiloxane capillary chromatography column. In some embodiments, the chromatography column used in the chromatography is an HP-5MS UI chromatography column. In some embodiments, the specification of the chromatography column is 30 m x 0.25 mm, 0.25 μm. In some embodiments, the chromatography column used in the chromatography is an HP-5MS UI chromatography column, 30 m x 0.25 mm, 0.25 μm.

[0034] In some embodiments, the chromatography adopts a temperature rising program.

[0035] In some embodiments, the starting temperature T1 of the temperature ramping procedure of the chromatography is 35-55 °C. In some embodiments, the starting temperature T1 is 35-45 °C. In some embodiments, the starting temperature T1 is about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 55 °C, or a range between any two of these values (including endpoints) or any value therein. In some embodiments, the starting temperature T1 is about 40 °C.

[0036] In some embodiments, the starting temperature hold time t1 of the temperature ramping procedure of the chromatography is 2-4 min. In some embodiments, the starting temperature hold time t1 is about 2 min, about 2.5 min, about 3 min, about 3.5 min, about 4 min, or a range between any two of these values (including endpoints) or any value therein. In some embodiments, the starting temperature hold time t1 is about 3 min.

[0037] In some embodiments, the temperature ramping rate v of the temperature ramping procedure of the chromatography is 15-25 °C / min. In some embodiments, the temperature ramping rate v is 18-22 °C / min. In some embodiments, the temperature ramping rate v is about 15 °C / min, about 16 °C / min, about 17 °C / min, about 18 °C / min, about 19 °C / min, about 20 °C / min, about 21 °C / min, about 22 °C / min, about 23 °C / min, about 24 °C / min, about 25 °C / min, or a range between any two of these values (including endpoints) or any value therein. In some embodiments, the temperature ramping rate v is about 20 °C / min.

[0038] In some embodiments, the ending temperature T2 of the temperature ramping procedure of the chromatography is 180-200 °C. In some embodiments, the ending temperature T2 is 185-195 °C. In some embodiments, the ending temperature T2 is about 180 °C, about 185 °C, about 190 °C, about 195 °C, about 200 °C, or a range between any two of these values (including endpoints) or any value therein. In some embodiments, the ending temperature T2 is about 190 °C.

[0039] In some embodiments, the hold time t2 at the termination temperature of the temperature ramp of the chromatography method is 3-10 min. In some embodiments, the hold time t2 at the termination temperature is 3-7 min. In some embodiments, the hold time t2 at the termination temperature is about 3 min, about 4 min, about 5 min, about 6 min, about 7 min, about 8 min, about 9 min, about 10 min, or a range between any two of these values, inclusive of the endpoints, or any value therein. In some embodiments, the hold time t2 at the termination temperature is about 5 min.

[0040] In some embodiments, the temperature ramp of the chromatography method is a temperature ramp of: a starting temperature of 35-55 °C held for 2-4 min, ramped to 180-200 °C at a rate of 15-25 °C / min, held for 3-10 min. In some embodiments, the temperature ramp of the chromatography method is a temperature ramp of: a starting temperature of 35-45 °C held for 2-4 min, ramped to 180-200 °C at a rate of 15-25 °C / min, held for 3-7 min. In some embodiments, the temperature ramp of the chromatography method is a temperature ramp of: a starting temperature of 40 °C held for 3 min, ramped to 190 °C at a rate of 20 °C / min, held for 5 min.

[0041] In some embodiments, the column flow of the chromatography method is 0.8-1.2 mL / min. In some embodiments, the column flow is 0.9-1.1 mL / min. In some embodiments, the column flow is about 0.8 mL / min, about 0.9 mL / min, about 1.0 mL / min, about 1.1 mL / min, about 1.2 mL / min, or a range between any two of these values, inclusive of the endpoints, or any value therein. In some embodiments, the column flow is about 1.0 mL / min.

[0042] In some embodiments, the injection volume of the chromatography method is 0.5-2.0 μL. In some embodiments, the injection volume is 0.5-1.5 μL. In some embodiments, the injection volume is 0.8-1.2 μL. In some embodiments, the injection volume is about 0.5 μL, about 0.8 μL, about 1.0 μL, about 1.2 μL, about 1.5 μL, about 2.0 μL, or a range between any two of these values, inclusive of the endpoints, or any value therein. In some embodiments, the injection volume is about 1.0 μL.

[0043] In some embodiments, the chromatography method is gas chromatography-mass spectrometry.

[0044] In some embodiments, the chromatographic conditions of the gas chromatography-mass spectrometry method further comprise one or more of the following:

[0045] Carrier gas: helium,

[0046] Column flow: 0.8-1.2 mL / min,

[0047] Injection volume: 0.5-2.0 μL,

[0048] Injection mode: splitless,

[0049] Injection port temperature: 200-260 °C.

[0050] In some embodiments, the chromatographic conditions of the gas chromatography-mass spectrometry method further comprise one or more of the following:

[0051] Carrier gas: helium,

[0052] Column flow: 1.0 mL / min,

[0053] Injection volume: 1.0 μL,

[0054] Injection mode: splitless,

[0055] Injection port temperature: 230 °C.

[0056] In some embodiments, the chromatographic conditions of the gas chromatography-mass spectrometry method further comprise:

[0057] Carrier gas: helium,

[0058] Column flow: 1.0 mL / min,

[0059] Injection volume: 1.0 μL,

[0060] Injection mode: splitless,

[0061] Injection port temperature: 230 °C.

[0062] In some embodiments, the mass spectrometry conditions of the gas chromatography-mass spectrometry method further comprise one or more of the following:

[0063] Ion source: electron impact (El) ionization source;

[0064] Ion source temperature: 220-240 °C,

[0065] Quadrupole rod temperature: 140-160 °C,

[0066] Transfer line temperature: 230-290 °C,

[0067] Scan mode: selected ion monitoring (SIM) mode,

[0068] Solvent delay time: 4-8 min,

[0069] Electron energy: 70 eV,

[0070] Incremental voltage: 50 V,

[0071] Quantitative ion: m / z 108,

[0072] Qualitative ion: m / z 79.

[0073] In some embodiments, the mass spectrometry conditions of the gas chromatography-mass spectrometry method further comprise one or more of the following:

[0074] Ion source: Electron impact (El) ionization source;

[0075] Ion source temperature: 230 °C,

[0076] Quadrupole rod temperature: 150 °C,

[0077] Transfer line temperature: 260 °C,

[0078] Scan mode: Selected ion monitoring (SIM) mode,

[0079] Solvent delay time: 6 min,

[0080] Electron energy: 70 eV,

[0081] Incremental voltage: 50 V,

[0082] Quantitative ion: m / z 108,

[0083] Qualitative ion: m / z 79.

[0084] In some embodiments, the mass spectrometry conditions of the gas chromatography-mass spectrometry method further comprise:

[0085] Ion source: Electron impact (El) ionization source,

[0086] Ion source temperature: 230 °C,

[0087] Quadrupole rod temperature: 150 °C,

[0088] Transfer line temperature: 260 °C,

[0089] Scan mode: Selected ion monitoring (SIM) mode,

[0090] Solvent delay time: 6 min,

[0091] Electron energy: 70 eV,

[0092] Incremental voltage: 50 V,

[0093] Quantitative ion: m / z 108,

[0094] Qualitative ions: m / z 79.

[0095] In some embodiments, the chromatographic conditions of the gas chromatography-mass spectrometry method are: the chromatographic column is an HP-5MS UI chromatographic column, 30 m x 0.25 mm, 0.25 μm, the temperature rising program is: the initial temperature is 40℃, maintained for 3 min, increased to 190℃ at a rate of 20℃ / min, maintained for 5 min, the carrier gas is helium, the column flow rate is 1.0 mL / min, the injection volume is 1.0 μL, the injection mode is splitless, the injection port temperature is 230℃; the mass spectrometric conditions of the gas chromatography-mass spectrometry method are: the ion source is an electron impact (EI) ionization source, the ion source temperature is 230℃, the quadrupole rod temperature is 150℃, the transfer line temperature is 260℃, the scan mode is a selective ion monitoring (SIM) mode, the solvent delay time is 6 min, the electron impact energy is 70 eV, the increment voltage is 50 V, the quantitative ion is m / z 108, and the qualitative ion is m / z 79.

[0096] In some embodiments, the present application provides a method for determining the residual amount of benzyl alcohol in a protein sample, comprising the following steps:

[0097] Step 1: according to the different protein concentrations in the sample, the sample is diluted by different multiples with ultrapure water, and the protein concentration after dilution is not higher than 40 mg / mL; if the protein concentration in the sample is not higher than 40 mg / mL, this step is omitted;

[0098] Specifically, when the protein concentration of the sample is > 40 mg / mL, an appropriate amount of sample is taken, an appropriate amount of ultrapure water is added, and vortex mixing is performed; if the dilution multiple is large, the process can be repeated for hierarchical dilution; the dilution multiple of each stage is not more than 20 times; the dilution multiple of this step is N; if the protein concentration of the sample is ≤ 40 mg / mL, ultrapure water is not added for dilution, and the polar organic solvent is directly added for extraction;

[0099] Step 2: the sample of step 1 is taken, a polar organic solvent is added, and the centrifuge tube is turned upside down several times; after the organic phase is taken out, the above operation can be optionally repeated, the organic phases are combined, and mixing is performed to obtain an extraction solution to be tested;

[0100] Specifically, the sample with a protein concentration not higher than 40 mg / mL is taken into a centrifuge tube, dichloromethane is added, the centrifuge tube is tightly capped, and the centrifuge tube is turned upside down several times; after the organic phase is taken out, an equal amount of dichloromethane is added, the centrifuge tube is tightly capped, and the centrifuge tube is turned upside down several times; after the organic phase is taken out, the organic phases of the two times are combined and mixed; the dilution multiple of this step is 2;

[0101] Step 3: the extraction solution is detected by gas chromatography-mass spectrometry, and a chromatogram is recorded;

[0102] Chromatographic conditions: the chromatographic column was HP-5MS UI chromatographic column, 30 m x 0.25 mm, 0.25 μm, the temperature program was: the initial temperature was 40℃, maintained for 3 min, increased to 190℃ at a rate of 20℃ / min, maintained for 5 min, carrier gas: helium, column flow rate: 1.0 mL / min, sample size: 1.0 μL, sample injection mode: splitless, sample injection port temperature: 230℃;

[0103] Mass spectrometric conditions: ion source: electron impact (EI) ion source, ion source temperature: 230℃, quadrupole temperature: 150℃, transfer line temperature: 260℃, scan mode: selective ion monitoring (SIM) mode, solvent delay time: 6 min, electron impact energy: 70 eV, increment voltage: 50 V, quantitative ion: m / z 108, qualitative ion: m / z 79;

[0104] Step 4, the concentration of benzyl alcohol in the extraction solution was calculated by external standard method, and the residual amount of benzyl alcohol in the sample was further calculated, and the specific calculation formula was as follows:

[0105] wherein,

[0106] C SA : measured value of the concentration of benzyl alcohol in the extraction solution (ng / mL);

[0107] C mAb : protein concentration in the sample (mg / mL);

[0108] DF: dilution factor of the extraction solution, determined by the actual preparation process of the solution.

[0109] The measured value of the concentration of benzyl alcohol in the extraction solution was calculated by standard curve method, including drawing a standard curve with the concentration of benzyl alcohol standard solution of different concentration gradients as the abscissa (X) and the corresponding peak area as the ordinate (Y), obtaining the linear regression equation, and substituting the peak area of benzyl alcohol in the extraction solution into the linear regression equation to calculate the measured value of the concentration of benzyl alcohol in the extraction solution (ng / mL).

[0110] In some embodiments, the concentration of the benzyl alcohol standard solution of different concentration gradients ranges from 14 to 160 ng / mL. In some embodiments, the concentrations of the benzyl alcohol standard solution of different concentration gradients are about 14, 40, 80, 128 and 160 ng / mL, respectively.

[0111] In some embodiments, the residual amount of benzyl alcohol detected by the method ranges from 0.7 to 8.0 ng / mg.

[0112] In some embodiments, the peak area, retention time, signal-to-noise ratio, etc. described in the method are derived from the detection results of the quantitative ion (m / z = 108).

[0113] In some embodiments, the present application provides the use of the method for determining benzyl alcohol residual amount in determining the benzyl alcohol residual amount of a process sample of a biological product or a bulk biological product.

[0114] The method for determining benzyl alcohol residual amount of the present application can effectively extract benzyl alcohol from a relatively complex sample system according to the physicochemical properties of benzyl alcohol and the matrix characteristics of the sample, and exclude the interference of other components, especially the interference of proteins, buffers and other formulation components, and can be widely used for determining the benzyl alcohol residual amount in biological products containing proteins, including process samples or bulk biological products. The determination method of the present application has the advantages of high extraction recovery, simple and economical operation, good detection specificity, low limit of quantification, good repeatability, high accuracy and precision, effectively reduces the interference of sample matrix, improves the detection accuracy and sensitivity of benzyl alcohol, and provides technical support for the quality control of process samples or bulk biological products. BRIEF DESCRIPTION OF DRAWINGS

[0115] Figure 1 is an extraction effect diagram of different extraction methods;

[0116] Figure 2 is a detection effect diagram of benzyl alcohol by different chromatographic columns; wherein a: the chromatogram corresponding to chromatographic column 1, b: the local enlarged diagram corresponding to chromatographic column 1, c: the chromatogram corresponding to chromatographic column 2, d: the local enlarged diagram corresponding to chromatographic column 2;

[0117] Figure 3 is a detection effect diagram of benzyl alcohol by different temperature programs;

[0118] Figure 4 is a system applicability test confirmation spectrum;

[0119] Figure 5 is a specificity test confirmation chromatogram;

[0120] Figure 6 is a chromatogram of the limit of quantification solution;

[0121] Figure 7 is a linear regression curve of benzyl alcohol;

[0122] Figure 8 is a column flow endurance chromatogram;

[0123] Figure 9 is an extraction frequency endurance chromatogram. DETAILED DESCRIPTION

[0124] The technical solutions of the present application are further illustrated below through specific examples, which do not represent limitation on the protection scope of the present application. Some non-essential modifications and adjustments made by others according to the concept of the present application still belong to the protection scope of the present application.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0126] "About" means a conventional error range that would be appreciated by one of ordinary skill in the art to which the corresponding value pertains. In some embodiments, "about" refers to ±10%, ±5%, ±1% of the stated value.

[0127] "Comprise" or "comprising" means including the elements listed thereafter without excluding other elements that can be included in the composition, method, etc. described, but does not exclude other elements that do not materially affect the combination. "Consisting essentially of means excluding other elements of any essential significance to the combination used for the intended purpose, but allowing for the presence of elements that do not materially affect the characteristics of the composition or method. "Consisting of means excluding elements not specifically listed. Embodiments defined by each of these transition terms are within the scope of the present application. For example, when a method is described as comprising steps A, B, and C, a method consisting essentially of steps A, B, and C, and a method consisting of steps A, B, and C are independently within the scope of the present application.

[0128] When the lower and upper limits of a range of values are disclosed, any value and any included range falling within that range are specifically disclosed. In particular, each range of values disclosed herein is to be understood to be individually defined and independently disclosed in its entirety in the disclosure of each and every stated value and range.

[0129] "Polar organic solvent" means an organic solvent that tends to interact with other compounds or itself through acid-base interactions, hydrogen bonding, dipole-dipole interactions, or through dipole-induced dipole interactions. Non-limiting examples of polar organic solvents include ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone; ethers such as tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, diisopropyl ether, or methyl tert-butyl ether; dimethylformamide; dimethylacetamide; dimethyl sulfoxide; acetonitrile; ethyl acetate; dichloromethane; N-methyl-2-pyrrolidone; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol, isobutanol, sec-butanol, or tert-butanol; and mixtures thereof.

[0130] “Chromatography” refers to an analytical technique in which a chemical mixture carried by a liquid or gas separates into its components as the mixture flows around or over a stationary liquid or solid phase due to differential distribution of the chemical entities. Non-limiting examples of chromatography, as used herein, include liquid chromatography (LC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), supercritical fluid chromatography (SFC), capillary electrophoresis chromatography (CEC).

[0131] “Gas chromatography” or “GC” refers to a chromatography in which a sample is evaporated and injected into a stream of carrier gas (such as nitrogen or helium) moving through a column containing a stationary phase composed of a liquid or a particulate solid and separated into its component compounds according to their affinity for the stationary phase. Both solvent and analyte are vaporized during injection due to the high temperature in the injection block and condense on the cooler column. As the column temperature is raised, the relatively less volatile analytes also enter the gas phase and eventually reach the detector. The more nonpolar, the more volatile the analyte, the more it is in affinity with the gas phase and the shorter the retention time. More polar or less volatile analytes are more in affinity with more polar stationary phases and reach the detector later. Possible detectors include flame ionization detector (FID), electron capture detector (ECD), nitrogen phosphorus detector (NPD), thermal conductivity detector (TCD), flame photometric detector (FPD), sulfur detector (SCD), mass spectrometer (MS).

[0132] “Mass spectrometry” or “MS” refers to an analytical technique by which compounds are identified by their mass, a method based on the filtering, detection, and measurement of ions by their mass-to-charge ratio (m / z). MS techniques generally include (1) ionization of a compound to form a charged compound; and (2) detection of the molecular weight of the charged compound and calculation of the mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A “mass spectrometer” generally includes an ionizer, a mass analyzer, and an ion detector. Typically, one or more molecules of interest are ionized, and the ions are then introduced into a mass analyzer, where, due to a combination of magnetic and electric fields, the ions follow paths in space that depend on mass (m) and charge (z) to separate, and the separated ions enter a detector, producing a signal.

[0133] “Permitted daily exposure” or “PDE” refers to the average maximum dose per day of a substance that is allowed to be ingested without producing toxicity. The PDE value for a particular substance is extrapolated from the no observed effect level, a body weight adjustment factor, a factor for interspecies differences, individual differences, a variable factor for acute toxicity studies for short-term exposure, and the like.

[0134] "Analytical Evaluation Threshold" or "AET" refers to the specific extractable and / or leachable content in each individual packaging container calculated according to the maximum allowable daily exposure or safety threshold / limiting threshold of a person, the dosage of the drug, and the characteristics of the packaging of the preparation, etc. When a specific extractable and / or leachable level reaches or exceeds this amount, the extractable / leachable needs to be analyzed and reported to the relevant department for safety evaluation. AET is the minimum concentration level in chemical analysis for determining whether a chemical substance needs to be evaluated for toxicology.

[0135] "Process sample" refers to the intermediate sample produced at the end of each interrelated operating unit in the production process of a biological product. The production process of a biological product is generally divided into an upstream process mainly based on cell culture (cell culture process) and a downstream process mainly based on multi-step purification procedures (purification process). The intermediate sample produced by the purification process is referred to as a purification process sample.

[0136] The downstream purification process of a protein-based biological product generally includes preliminary purification, intermediate purification, fine purification, ultrafiltration concentration and replacement, etc. "Preliminary purification" is a purification step aimed at capturing target proteins, usually at the initial step of the downstream purification process, which can remove most of the process-related impurities. Common preliminary purification methods include protein A affinity chromatography, ion exchange chromatography. "Intermediate purification" refers to the step for further removing process-related impurities (host proteins HCP, DNA, etc.), which is at the intermediate step of the downstream purification process. Common intermediate purification methods include anion exchange chromatography or composite mode chromatography. "Fine purification" is aimed at further purifying target proteins and removing product-related impurities (such as aggregates, fragments, charge isoforms), which is at the terminal step of the downstream purification process. Common fine purification methods include cation exchange chromatography, hydrophobic chromatography, composite mode chromatography or hydroxyapatite chromatography. "Ultrafiltration" is the use of ultrafiltration membrane stacks with pore sizes suitable for the molecular weight of the product to replace and concentrate the protein buffer, so that the sample properties meet the formulation requirements. The membrane material of the ultrafiltration membrane is usually PES (polyether sulfone) or modified or unmodified regenerated cellulose.

[0137] A "protein" refers to an organic compound composed of a linear chain of two or more amino acids. The compound can have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids; two hundred or more amino acids; or even three hundred or more amino acids. The term "protein" as used herein refers to molecules having the sequence of a native protein (i.e., a protein produced by a naturally occurring, particularly non-recombinant, cell or a genetically engineered or recombinant cell) and includes molecules having the amino acid sequence of the native protein or molecule with one or more deletions, additions, and / or substitutions of amino acids having the native sequence. In some embodiments, the protein to be purified is an antibody. As contemplated herein, the term "protein" includes "polypeptide," "immunoglobulin," "antibody," and "antibody fragment."

[0138] An "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be an intact antibody and any antigen-binding fragment thereof or a single chain thereof. Thus, the term "antibody" includes any protein or peptide that comprises at least a portion of an immunoglobulin molecule that has a biological activity characteristic of an antigen-binding molecule. Examples of such include, but are not limited to, a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein. An antibody can specifically recognize and bind one or more (e.g., two) antigens. An antibody that specifically recognizes and binds multiple (e.g., two) antigens can be referred to as a multispecific (e.g., bispecific) antibody.

[0139] "Antibodies" include a wide variety of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that the class of a heavy chain includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses among them (e.g., γ1-γ4). The "class" of an antibody is determined by the nature of the constant domain of the heavy chain. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and the functional specificities conferred are known.

[0140] Antibodies or derivatives thereof include, but are not limited to, monoclonal, polyclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), fragments containing either a VL or VH domain, or fragments produced using a Fab expression library, and anti-idiotypic (anti-Id) antibodies. "Antibodies" are also intended to refer to modified products of antibodies, including but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. In some embodiments, antibodies include glycosylation modifications.

[0141] Antibodies can be derived from any animal, including birds and mammals, such as antibodies of human, murine, equine, rabbit, goat, camel, llama, horse, chicken, or chondrichthyan origin (e.g., from a shark).

[0142] "Antibody drug conjugate" or "ADC" refers to a polypeptide (such as an antibody or antigen-binding unit thereof) that binds a target of interest linked to one or more chemical drugs, which can optionally be a therapeutic or cytotoxic agent. In some embodiments, an ADC includes an antibody, a drug (e.g., a cytotoxic drug), and a linker capable of attaching or conjugating the drug to the antibody. Non-limiting examples of drugs that can be included in an ADC are mitotic inhibitors, antitumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormones, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors (e.g., TEC-family kinase inhibitors and serine / threonine kinase inhibitors), and radiosensitizers.

[0143] The antibody BAT4306F is the antibody BAT4306F disclosed in patent document WO 2019 / 029713 A1.

[0144] Unless otherwise specified, the instruments and equipment, reagents and samples, solution preparation, detection methods, and the like used in the specific embodiments of the present application are as follows:

[0145] 1. Instruments and equipment:

[0146] Gas chromatograph-mass spectrometer: Agilent 8890-5977B;

[0147] Chromatographic column 1: Agilent HP-5MS UI (30 m x 0.25 mm, 0.25 μm);

[0148] Column 2: Agilent DB-WAX (30 m x 0.32 mm, 0.5 μm).

[0149] 2. Reagents and samples:

[0150] 2.1 Reagents

[0151] Dichloromethane (DCM), chromatographic grade;

[0152] Acetonitrile (ACN), chromatographic grade;

[0153] Ethyl acetate (EA), chromatographic grade;

[0154] n-Hexane (Hex), chromatographic grade;

[0155] Acetone (AC), chromatographic grade;

[0156] Water (H2O), ultrapure water.

[0157] 2.2 Standard

[0158] Benzylic alcohol (BnOH), 99.9% purity, purchased from Shanghai Anpu;

[0159] 2.3 Test samples:

[0160] Table 1a Test sample information

[0161] Table 1b Test sample information

[0162] Note: The above test samples are obtained by self-made.

[0163] 3. Solution preparation

[0164] 3.1 Blank solution (RB): DCM.

[0165] 3.2 Method blank solution (MB), buffer blank solution (FB)

[0166] MB: Take 500 μL ultrapure water into a 2 mL centrifuge tube, add 500 μL DCM, tightly cover the centrifuge tube, and invert it ten times, centrifuge at 8000 rpm for 3 min, take out the lower organic phase, add 500 μL DCM again, tightly cover the centrifuge tube, and invert it ten times, centrifuge at 8000 rpm for 3 min, take out the lower organic phase, combine the two times of organic phase, mix well, and obtain the method blank solution.

[0167] FB: Take 500 μL buffer into a 2 mL centrifuge tube, add 500 μL DCM, cover the centrifuge tube tightly, turn it upside down for 10 times, centrifuge at 8000 rpm for 3 min, take out the lower organic phase, add 500 μL DCM again, cover the centrifuge tube tightly, turn it upside down for 10 times, centrifuge at 8000 rpm for 3 min, take out the lower organic phase, combine the two organic phases, mix well, and the buffer blank solution is obtained.

[0168] 3.3 Preparation of benzyl alcohol linear solution

[0169] An appropriate amount of benzyl alcohol stock solution (1622.4 ng / mL) was taken and diluted into different concentration gradients of benzyl alcohol linear solution (concentrations of about 14, 40, 80, 128, and 160 ng / mL) according to the method shown in Table 2.

[0170] Table 2 Preparation method of linear solution

[0171] 3.4 Calibration control solution (VS) and system suitability solution (SST)

[0172] VS: 1 portion was prepared in the same way as Line-3, and the VS corresponded to different benzyl alcohol stock solutions than the linear solution. The first needle VS was used as the system suitability solution (SST).

[0173] 3.5 Test sample solution (SA)

[0174] 3.5.1 When the concentration of test sample protein is ≤40 mg / mL: 500 μL sample was taken into a 2 mL centrifuge tube, 500 μL DCM was added, the centrifuge tube was covered tightly, and it was turned upside down for 10 times, centrifuged at 8000 rpm for 3 min, the lower organic phase was taken out, 500 μL DCM was added again, the centrifuge tube was covered tightly, and it was turned upside down for 10 times, centrifuged at 8000 rpm for 3 min, the lower organic phase was taken out, and the two organic phases were combined and mixed well to obtain the test sample solution. At this time, the dilution factor of the test sample solution was 2.

[0175] 3.5.2 When the concentration of test sample protein is >40 mg / mL: an appropriate amount of sample (≥50 μL) was taken, an appropriate amount of ultrapure water was added (total volume ≥500 μL), and vortexed to mix well. If the dilution factor is large, the process can be repeated for hierarchical dilution. The dilution factor of each level should not exceed 20. The dilution factor of this step was N. 500 μL of the diluted sample was taken into a 2 mL centrifuge tube, 500 μL DCM was added, the centrifuge tube was covered tightly, and it was turned upside down for 10 times, centrifuged at 8000 rpm for 3 min, the lower organic phase was taken out, 500 μL DCM was added again, the centrifuge tube was covered tightly, and it was turned upside down for 10 times, centrifuged at 8000 rpm for 3 min, the lower organic phase was taken out, and the two organic phases were combined and mixed well to obtain the test sample solution. The dilution factor of this step was 2. At this time, the dilution factor of the test sample solution was 2N.

[0176] 3.6 Spiked solution (SP)

[0177] 3.6.1 Spiked solution of test sample

[0178] According to the preparation method of the test sample solution, 500 μL of sample or diluted sample was taken into a 2 mL centrifuge tube, 20 μL of 4 μg / mL benzyl alcohol standard solution was added, mixed, 500 μL of DCM was added, the centrifuge tube was tightly covered, and each of the positive and negative was inverted for ten times, centrifuged at 8000 rpm for 3 min, the lower organic phase was taken out, 500 μL of DCM was added again, the centrifuge tube was tightly covered, each of the positive and negative was inverted for ten times, centrifuged at 8000 rpm for 3 min, the lower organic phase was taken out, and the organic phases of the two times were combined and mixed to obtain the solution.

[0179] 3.6.2 Buffer spiked solution

[0180] 3.6.2.1 UF buffer low concentration spiked solution of tocilizumab and BAT4306F (UF buffer_LSP)

[0181] 500 μL of UF buffer was taken into a 2 mL centrifuge tube, 20 μL of 672 ng / mL benzyl alcohol standard solution was added, mixed, 500 μL of DCM was added, the centrifuge tube was tightly covered, each of the positive and negative was inverted for ten times, centrifuged at 8000 rpm for 3 min, the lower organic phase was taken out, 500 μL of DCM was added again, the centrifuge tube was tightly covered, each of the positive and negative was inverted for ten times, centrifuged at 8000 rpm for 3 min, the lower organic phase was taken out, and the organic phases of the two times were combined and mixed to obtain the solution.

[0182] 3.6.2.2 UF buffer medium concentration spiked solution of bevacizumab and secukinumab (UF buffer_MSP)

[0183] 500 μL of UF buffer was taken into a 2 mL centrifuge tube, 20 μL of 4 μg / mL benzyl alcohol standard solution was added, mixed, 500 μL of DCM was added, the centrifuge tube was tightly covered, each of the positive and negative was inverted for ten times, centrifuged at 8000 rpm for 3 min, the lower organic phase was taken out, 500 μL of DCM was added again, the centrifuge tube was tightly covered, each of the positive and negative was inverted for ten times, centrifuged at 8000 rpm for 3 min, the lower organic phase was taken out, and the organic phases of the two times were combined and mixed to obtain the solution.

[0184] 3.6.2.3 UF buffer high concentration spiked solution of dupilumab (UF buffer_HSP)

[0185] Take 500 μL UF buffer into a 2 mL centrifuge tube, add 20 μL of 8 μg / mL benzyl alcohol standard solution, mix well, add 500 μL DCM, cover the centrifuge tube tightly, turn it upside down for ten times, centrifuge at 8000 rpm for 3 min, take out the lower organic phase, add another 500 μL DCM, cover the centrifuge tube tightly, turn it upside down for ten times, centrifuge at 8000 rpm for 3 min, take out the lower organic phase, combine the two organic phases, mix well to obtain.

[0186] 4. Detection method

[0187] 4.1 Chromatographic conditions

[0188] Chromatographic column: HP-5MS UI (30 m x 0.25 mm, 0.25 μm),

[0189] Temperature program: initial temperature 40℃, hold for 3 min, increase to 190℃ at a rate of 20℃ / min, hold for 5 min,

[0190] Carrier gas: helium,

[0191] Column flow rate: 1.0 mL / min,

[0192] Injection volume: 1.0 μL,

[0193] Injection mode: splitless,

[0194] Injection port temperature: 230℃;

[0195] 4.2 Mass spectrometric conditions

[0196] Ion source: electron impact (EI) ionization source,

[0197] Ion source temperature: 230℃,

[0198] Quadrupole rod temperature: 150℃,

[0199] Transfer line temperature: 260℃,

[0200] Scan mode: selective ion monitoring (SIM) mode,

[0201] Solvent delay time: 6 min,

[0202] Electron impact energy: 70 eV,

[0203] Incremental voltage: 50 V,

[0204] Quantitative ion: m / z 108,

[0205] Qualitative ion: m / z 79.

[0206] 5. Calculation method

[0207] The standard curve was plotted with the concentration of benzyl alcohol as the horizontal coordinate (X) and the corresponding peak area as the vertical coordinate (Y) by external standard method. The linear regression equation was obtained. The peak area of benzyl alcohol in the extraction solution was substituted into the linear regression equation to calculate the measured value of the concentration of benzyl alcohol in the extraction solution (ng / mL). The linear regression equation and the concentration of benzyl alcohol were automatically calculated by the instrument software.

[0208] Calculation formula:

[0209] In the formula:

[0210] C SP : measured value of the concentration of benzyl alcohol in the test sample solution (ng / mL);

[0211] C SA : measured value of the concentration of benzyl alcohol in the test sample solution (ng / mL);

[0212] C TH : theoretical added concentration of benzyl alcohol in the test sample solution (ng / mL);

[0213] C mAb : concentration of protein in the sample (mg / mL);

[0214] DF: dilution multiple of the test sample solution, determined by the actual preparation process of the solution;

[0215] Note: The data related to the quantitative results described in the present application, such as peak area, tailing factor and signal-to-noise ratio, should be derived from the detection results of the quantitative ion (m / z = 108).

[0216] In the following examples, unless otherwise specified, the reagents and instruments used are conventional reagents and instruments in the art, which can be obtained by commercial means; the methods used are conventional technical methods in the art, and those skilled in the art can implement the methods and obtain the corresponding results without any doubt according to the content of the examples.

[0217] Example 1: Screening and optimization of extraction solvent

[0218] Referring to the preparation method under item “3.6.2.2”, the effects of dichloromethane, acetonitrile, ethyl acetate and n-hexane as extraction solvents on the extraction efficiency of benzyl alcohol in the test sample solution of ustekinumab P(P) Buffer were investigated. Each extraction solution was detected by the detection method under items “4.1-4.2”. The detection results are shown in Table 3.

[0219] Table 3: Extraction efficiency of different extraction solvents

[0220] The results show that the recovery rate is only 18.9% when n-hexane is used as the extraction solvent, and the extraction efficiency is poor; the recovery rates of dichloromethane, acetonitrile and ethyl acetate are all between 90% and 110%, and the extraction efficiency of dichloromethane is the best. Considering that acetonitrile is miscible with water and a large amount of salt needs to be added to promote layering, and that the boiling point of ethyl acetate is relatively low and the density is smaller than that of water, the ethyl acetate phase is located in the upper layer after layering, which is more volatile. Therefore, dichloromethane is selected as the extraction solvent for subsequent experiments.

[0221] Example 2: Screening and optimization of extraction methods

[0222] Referring to the preparation method under item “3.5.1”, the effects of two different extraction methods, i.e., forward and reverse inversion and vortex, on the extraction efficiency were investigated by taking tocilizumab UF sample as the test sample in the process of extracting benzyl alcohol.

[0223] As shown in FIG. 1, when the forward and reverse inversion extraction method is adopted, two phases can be seen after centrifugation, the boundary is clear, and the solution is clear. When the vortex extraction method is adopted, the solution shows obvious emulsification, and the emulsion cannot be eliminated even by increasing ultrasonic treatment. Therefore, the forward and reverse inversion extraction method is selected for subsequent experiments.

[0224] Example 3: Screening and optimization of chromatographic columns

[0225] The properties of the chromatographic column packing are an important factor affecting the detection effect. Therefore, referring to the preparation method under item “3.3”, 100 ng / mL benzyl alcohol standard solution was prepared and injected for detection, and the following temperature rising program was adopted: the initial temperature was 40°C, which was increased to 220°C at a rate of 20°C / min, and maintained for 5 min, and the FID detector recorded the signal, and other chromatographic conditions were the same as those specified under item “4.1”. The detection effects of different chromatographic columns (chromatographic column 1 and chromatographic column 2) on benzyl alcohol were investigated. The detection results are shown in FIG. 2.

[0226] The results show that under the same concentration, the response signal-to-noise ratios of benzyl alcohol detected by chromatographic column 1 and chromatographic column 2 are 30.2 and 4.0, respectively, and it can be seen that the detection sensitivity of chromatographic column 1 for benzyl alcohol is significantly higher than that of chromatographic column 2. Therefore, in order to obtain higher response, chromatographic column 1 is selected for subsequent experiments.

[0227] Example 4: Screening and optimization of temperature rising programs

[0228] The column temperature of the chromatographic column has a significant effect on the detection effect. Therefore, referring to the preparation method under item “3.3”, 100 ng / mL benzyl alcohol standard solution was prepared and injected for detection under the chromatographic conditions of item “4.1” using an FID detector, and the detection effects of different temperature rising programs shown in Table 4 on benzyl alcohol were investigated. The detection results are shown in FIG. 3 and Table 5.

[0229] Table 4 Different temperature program settings

[0230] Table 5 Column efficiency data of different temperature programs

[0231] The results show that the No. 1-6 temperature programs can achieve effective separation and detection of benzyl alcohol, the peak tailing factor of benzyl alcohol of No. 3 and No. 4 temperature programs is relatively large, the theoretical plate number of benzyl alcohol of No. 5 and No. 6 temperature programs is relatively low, while the peak shape of benzyl alcohol of No. 1 and No. 2 temperature programs is good, and the retention time is moderate. Considering that the total running time of No. 1 temperature program is shorter, therefore, No. 1 temperature program is selected for other subsequent tests.

[0232] Methodology validation of Example 5

[0233] System suitability

[0234] Prepare 1 portion of system suitability solution, and divide it into 6 injection bottles for continuous injection detection. The confirmation results are shown in Figure 4 and Table 6, the peak area RSD of benzyl alcohol in the 6 needle system suitability solution is 2.1%, the RSD of retention time is 0.1%, the tailing factor is between 1.3-1.4, and the theoretical plate number is between 673944-729069. It can be seen that the system suitability of this method is good.

[0235] Table 6 Confirmation results of system suitability

[0236] Specificity test

[0237] Prepare 1 portion of blank solution (RB), method blank solution (MB), system suitability solution (SST), P (P) buffer blank solution of ustekinumab (FB), P (P) test solution of ustekinumab and its test solution with standard addition (P(P)_SP), UF buffer blank solution of golimumab (FB), UF test solution of golimumab and its test solution with standard addition (UF_SP), 1 portion of UF buffer blank solution of bevacizumab, tocilizumab, secukinumab, dupilumab and BAT4306F and its UF buffer solution with standard addition (UF buffer_LSP, UF buffer_MSP, UF buffer_HSP), and 1 portion of control solution with similar concentration level according to the standard addition concentration (Line-1 (LOQ), Line-3, Line-5). Each injection is 1 needle, and the chromatogram is recorded for specificity analysis. The specific results are shown in Figure 5 and Table 8.

[0238] The results show that the RB and MB do not interfere with the peak of benzyl alcohol. In the FB solution of golimumab, the peak area of the interfering peak is 28% of the limit of quantification (LOQ), which is not higher than 30% of the LOQ, meeting the acceptable standard; the peaks of the other FB do not interfere with the peak of benzyl alcohol. The peak of benzyl alcohol in the chromatogram of the test sample spiked solution does not interfere, and the peak area of benzyl alcohol at the retention time is larger than that of the test sample solution. The peak of benzyl alcohol in the chromatogram of the buffer spiked solution does not interfere, and the peak area of benzyl alcohol at the retention time is larger than that of the FB. The deviation of the relative ion intensity of the qualitative ion / quantitative ion of the test sample spiked solution and the buffer spiked solution from the control solution at a similar concentration level meets the requirements of Table 7. It can be seen that the specificity of the method is good.

[0239] Table 7: Relative ion intensity requirements

[0240] Table 8: Confirmation results of relative ion intensity

[0241] Limit of quantification

[0242] The benzyl alcohol stock solution (1622.4 ng / mL) was diluted with DCM to a certain concentration. When the signal-to-noise ratio (S / N) was ≥20, and the spiked solution at this concentration met the accuracy and repeatability requirements, it was the limit of quantification solution (LOQ). Six LOQ solutions were prepared in parallel, each was injected once, and the chromatogram was recorded. The specific results are shown in Figure 6.

[0243] The results show that the minimum S / N of the benzyl alcohol peak in the 6 LOQ solutions is 114, and the peak area RSD is 1.7%. The corresponding concentration is 13.63 ng / mL, which is less than 20% (15.7 ng / mL) of the analysis evaluation threshold AET (78.3 ng / mL) of benzyl alcohol. According to the repeatability and accuracy confirmation results, the spiked solution at the LOQ concentration meets the accuracy and repeatability requirements. Therefore, the limit of quantification of this method is determined to be 14 ng / mL.

[0244] Linearity and range

[0245] The benzyl alcohol standard solution was prepared by the method under item “3.3”, and each was injected once, and the chromatogram was recorded. The standard curve (see Figure 7) was drawn with the benzyl alcohol concentration of the benzyl alcohol standard solution at different concentration gradients as the abscissa (X) and the corresponding peak area as the ordinate (Y). The linear regression equation is y = 307.36821x + 765.47185, R 2 = 0.9974. The linear regression equation, correlation coefficient, and measured concentration are automatically calculated by the instrument software.

[0246] It can be seen that benzyl alcohol has good linearity in the LOQ~200% limit range, and the linear range is 13.63 ng / mL~162.24 ng / mL.

[0247] Precision

[0248] 1) Repeatability

[0249] According to the preparation method under items "3.5" and "3.6.1", two P(P) test sample solutions of ustekinumab and six P(P) test sample spiked solutions of ustekinumab were independently prepared by the same analyst in parallel. Each sample was injected once, and the chromatogram was recorded. After deducting the content of benzyl alcohol in the test sample solution, the benzyl alcohol spiked recovery rate of the six P(P) test sample spiked solutions was calculated.

[0250] The results are shown in Table 9. The spiked recovery rate of benzyl alcohol in the six P(P) test sample spiked solutions was between 94.4% and 108.6%, and the RSD was 5.8%. This indicates that the repeatability of the method is good.

[0251] Table 9 Confirmation results of repeatability Note: ND indicates not detected.

[0252] 2) Intermediate precision

[0253] The above operation under "Repeatability" was repeated by another analyst independently on different dates using different batches of chromatographic columns.

[0254] The results are shown in Table 10. The spiked recovery rate of benzyl alcohol in the six P(P) test sample spiked solutions was between 108.7% and 120.7%, and the RSD was 4.0%. The RSD of the spiked recovery rate of benzyl alcohol in the twelve P(P) test sample spiked solutions of two analysts was 9.0%. This indicates that the intermediate precision of the method is good.

[0255] Table 10 Confirmation results of intermediate precision Note: ND indicates not detected.

[0256] Accuracy

[0257] 1) Test sample spiking

[0258] Take 500 μL of the P(P) sample of ustekinumab into a 2 mL centrifuge tube, add 20 μL of 672 ng / mL, 4 μg / mL, and 8 μg / mL of the benzyl alcohol standard solution respectively, and prepare the test sample spiked solutions (P(P)_LSP, P(P)_MSP, P(P)_HSP) of low, medium, and high concentration levels respectively according to the preparation method under item “3.6.1”. Prepare 3 replicates of each concentration level of the spiked solution. Take the above 9 accuracy solutions, each inject 1 needle, and record the chromatogram; deduct the benzyl alcohol content of the test sample solution under item “Step 1) repeatability”, and calculate the benzyl alcohol spiked recovery rate of the 9 accuracy solutions. The results are shown in Table 11.

[0259] Table 11 Accuracy confirmation results of ustekinumab test sample spiking Note: ND indicates not detected.

[0260] Take 500 μL of the P(P) sample of ustekinumab into a 2 mL centrifuge tube, add 20 μL of 672 ng / mL, 4 μg / mL, and 8 μg / mL of the benzyl alcohol standard solution respectively, and prepare the test sample spiked solutions (P(P)_LSP, P(P)_MSP, P(P)_HSP) of low, medium, and high concentration levels respectively according to the preparation method under item “3.6.1”. Prepare 3 replicates of each concentration level of the spiked solution. Take the above 9 accuracy solutions, each inject 1 needle, and record the chromatogram; deduct the benzyl alcohol content of the test sample solution under item “Step 1) repeatability”, and calculate the benzyl alcohol spiked recovery rate of the 9 accuracy solutions. The results are shown in Table 11.

[0261] Table 11 Accuracy confirmation results of ustekinumab test sample spiking Note: ND indicates not detected.

[0262] 2) Buffer spiking

[0263] Prepare 1 portion of the UF buffer blank solution of tocilizumab, BAT4306F, bevacizumab, secukinumab, and dupilumab, and 2 portions of the corresponding UF buffer spiked solution (i.e. accuracy solution) according to the preparation method under items “3.2” and “3.6.2”. Inject 1 needle each, and record the chromatogram; deduct the benzyl alcohol content of each UF buffer blank solution, and calculate the benzyl alcohol spiked recovery rate of the 10 accuracy solutions. The results are shown in Table 13.

[0264] Table 13 Accuracy confirmation results of buffer spiking Note: ND indicates not detected.

[0265] From the data in Tables 11-13, it can be seen that the accuracy of the method is good.

[0266] Durability

[0267] The durability test projects are column flow, extraction times, and sample solution stability.

[0268] 1) Column flow durability

[0269] Prepare RB, SST, each linear solution, and two portions of the P(P) sample spiked solution of ustekinumab. Use different column flow (0.9 mL / min, 1.0 mL / min, 1.1 mL / min), and other conditions are the same as the detection method under item “4.1”. Record the chromatogram (see Figure 8), and calculate the benzyl alcohol spike recovery rate of the spiked solution.

[0270] The column flow durability test results show that the system suitability meets the requirements, the benzyl alcohol spike recovery rate is between 91.3% and 99.6%, and the RSD is 3.3%. This indicates that the method has good column flow durability.

[0271] 2) Extraction times durability

[0272] Prepare two portions of the P(P) sample spiked solution of ustekinumab, and add 500 μL of DCM to the remaining aqueous phase after the P(P) sample spiked solution is prepared. Tighten the centrifuge tube, and turn it over ten times in both directions. Centrifuge at 8000 rpm for 3 min, and take out the lower organic phase. Add 500 μL of DCM for dilution to obtain the third extraction solution. Detect each solution by the detection method under item “4.1”, record the chromatogram (see Figure 9), and calculate the benzyl alcohol spike recovery rate of the spiked solution.

[0273] The extraction times durability test results show that the ratio of the peak area of benzyl alcohol in the third extraction solution to the peak area of benzyl alcohol in the spiked solution is ≤5.7% when comparing the two portions of the third extraction solution with the corresponding spiked solution. This indicates that the method has good extraction times durability.

[0274] 3) Solution stability

[0275] Prepare six portions of the P(P) sample spiked solution of ustekinumab, mix well, and then load into the sample vial. Detect at different time points from 0 to 24 h by the detection method under item “4.1”, and calculate the benzyl alcohol spike recovery rate of the spiked solution.

[0276] The solution stability results are shown in Table 14, the benzyl alcohol spike recovery rate is between 101.2% and 117.7%, and the RSD is 5.5%. This indicates that the method has good solution stability.

[0277] Table 14 Confirmation results of sample solution stability

[0278] Example 6 Determination of benzyl alcohol residual amount

[0279] Refer to the preparation method under item "3.5", prepare the test solution of P(C), P(M), P(P), UF, DS of ustekinumab and golimumab respectively, and prepare two parallel samples. Take 1 needle of each of the above test solution as sample, and record the chromatogram according to the detection method under items "4.1-4.2". If the detection result is lower than LOQ, report "not detected", and report the detected concentration if it is higher than LOQ.

[0280] The results are shown in Table 15. The detected concentration of benzyl alcohol in the P(C) test sample of ustekinumab is 17.38 ng / mL, and benzyl alcohol is not detected in the P(M), P(P), UF, and DS test samples. The detected concentration of benzyl alcohol in the P(M) test sample of golimumab is 30.16 ng / mL, and benzyl alcohol is not detected in the P(C), P(P), UF, and DS test samples.

[0281] Table 15 Determination results of benzyl alcohol residual amount in samples Note: ND indicates not detected.

Claims

1. A method of determining the residual amount of benzyl alcohol in a sample comprising: The benzyl alcohol in the sample is extracted by a polar organic solvent, and the residual amount of benzyl alcohol in the extracted solution is determined by chromatography, wherein the sample is a biological product sample.

2. The method of claim 1, wherein the sample is a process sample of a biological product.

3. The method of claim 1, wherein the sample is a bulk solution of a biological product.

4. The method of any one of claims 1-3, wherein the process is a purification process.

5. The method of any one of claims 1-4, wherein the process is selected from one or more of preliminary purification, intermediate purification, fine purification, and ultrafiltration.

6. The method of any one of claims 1-5, wherein the biological product is a protein-containing biological product.

7. The method of any one of claims 1-6, wherein the biological product is selected from one or more of a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a recombinant protein, a tagged fusion protein, and an antibody drug conjugate product.

8. The method of any one of claims 1-7, wherein the biological product is selected from one or more of a chimeric antibody, a murine antibody, a primatized antibody, a humanized antibody, or a fully human antibody.

9. The method of any one of claims 1-8, wherein the biological product is selected from one or more of ustekinumab, golimumab, tocilizumab, bevacizumab, dupilumab, or BAT4306F.

10. The method of any one of claims 1-9, wherein the operation of extracting the benzyl alcohol in the sample by a polar organic solvent is preceded by diluting the sample with ultrapure water by different multiples according to the protein concentration in the sample, and the protein concentration after dilution is not higher than 40 mg / mL; if the protein concentration in the sample is not higher than 40 mg / mL, this step is omitted.

11. The method of any one of claims 1-10, comprising the following steps: Step 1, diluting the sample with ultrapure water by different multiples according to the protein concentration in the sample, and the protein concentration after dilution is not higher than 40 mg / mL; if the protein concentration in the sample is not higher than 40 mg / mL, this step is omitted; Step 2, taking the sample of step 1, adding a polar organic solvent, inverting the sample several times, centrifuging, and taking out the organic phase, and optionally repeating the above operation, combining the organic phases, and mixing to obtain an extracted solution to be tested; Step 3, detecting the extracted solution by chromatography, and recording the chromatogram; Step 4, the concentration of benzyl alcohol in the extraction solution was calculated by external standard method, and the residual amount of benzyl alcohol in the sample was further calculated, and the specific calculation formula was as follows: wherein, C SA : measured value of benzyl alcohol concentration in the extraction solution (ng / mL); C mAb : Protein concentration in sample (mg / mL); DF: dilution multiple of the extracted solution, determined by the actual preparation process of the solution.

12. The method of any one of claims 1-11, wherein the polar organic solvent is dichloromethane, ethyl acetate, or acetonitrile.

13. The method of any one of claims 1-12, wherein the chromatography employs a non-polar or weakly polar chromatographic column.

14. The method of any one of claims 1-13, wherein the chromatography employs a non-polar or weakly polar polysiloxane capillary chromatographic column.

15. The method of any one of claims 1-14, wherein the chromatography employs a (5%-phenyl)-methyl polysiloxane capillary chromatographic column.

16. The method of any one of claims 1-15, wherein the chromatography employs a HP-5MS UI column.

17. The method of any one of claims 1-16, wherein the column specifications are 30 m x 0.25 mm, 0.25 μιη.

18. The method of any one of claims 1-17, wherein the chromatography employs a temperature program of 35-55 °C for 2-4 min, ramped at 15-25 °C / min to 180-200 °C, and held for 3-10 min.

19. The method of any one of claims 1-18, wherein the chromatography is gas chromatography-mass spectrometry.

20. The method of claim 19, wherein the chromatography conditions of the gas chromatography-mass spectrometry further comprise one or more of the following: carrier gas: helium, column flow: 0.8-1.2 mL / min, injection volume: 0.5-2.0 μΐ, injection mode: splitless, injection port temperature: 200-260 °C.

21. The method of claim 19 or 20, wherein the mass spectrometry conditions of the gas chromatography-mass spectrometry further comprise one or more of the following: ion source: electron impact (El) ionization source, ion source temperature: 220-240 °C, quadrupole rod temperature: 140-160 °C, transfer line temperature: 230-290 °C, scan mode: selected ion monitoring (SIM) mode, solvent delay time: 4-8 min, electron impact energy: 70 eV, incremental voltage: 50 V, quantitation ion: m / z 108, qualitative ion: m / z 79.

22. Use of the method of any one of claims 1-21 for determining benzyl alcohol residual amount in a process sample of a biological product or in a bulk biological product.

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